Treatment protocol

ABSTRACT

Combination treatments with an antiangiogenesis agent and non-toxic blood levels of ethanol and/or DMSO are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. application Ser. No.12/994,105 having an international filing date of 22 May 2009, which isthe national phase of PCT application PCT/US2009/045062 having aninternational filing date of 22 May 2009, which claims benefit under 35U.S.C. §119(e) to provisional U.S. application No. 61/055,413 filed 22May 2008. The contents of the above patent applications are incorporatedby reference herein in their entirety.

TECHNICAL FIELD

The invention relates to diagnostic tests useful in evaluative screeningof subjects for general wellbeing. More particularly, the invention isdirected to an inexpensive and convenient assay for circulatingendothelial cells and for detection of endothelial cells in general. Inaddition, the invention relates to an improved protocol for treatment ofconditions characterized by unwanted neovasculature, includingneoplastic conditions.

BACKGROUND ART

It is generally recognized that circulating microvascular (endothelial)cells are important markers for a variety of disease states such ascardiac disease, cardiovascular disease and diabetes (Bos, C. J., etal., J. Am. Col. Cardiol. (2006) 48:1538-1547). Detecting such cells inthe circulatory system is, however, problematic because of interferencefrom other cells, such as platelets. Methods that involve separation ofcells by type, or antibody labeling such as the use of antibodiesdirected to CD31, require time-consuming steps and often requireexpensive equipment.

PCT application US 2006/047954, published as WO 2007/075440 andincorporated herein by reference, which describes the work of theapplicant herein, discloses a method for assessing the effect of drugsused in antineoplastic therapy in microaggregates of tumor biopsies byfirst identifying, through labeling with CD31 or through experiencedobservation, endothelial cells as opposed to neoplastic cells in thesample. The sample, after being subjected to a candidate drug, isstained with a dye which is taken up by dead cells but excluded byviable cells and then counterstained with a dye that is accepted byviable cells. When the sample is examined under a microscope for theparticular dye combination selected, the blue/green dye which isexcluded by viable cells shows the dead cells as “blueberries” in abackground pink “pancake” of viable cells stained with the counterstain.It is possible, therefore, by virtue of the knowledge of which cells arecancer cells and which cells are endothelial cells to determine whethera given drug affects the cancer cells, endothelial cells or both. Ingeneral, it is shown that drugs known to inhibit angiogenesis, such asAvastin® (bevacizumab), result in the endothelial cells showing up deadin this assay while the tumor cells remain unaffected. Directantineoplastic drugs result in the death of the tumor cells themselves,rather than the endothelial cells.

It has now been found that endothelial cells, when caused to becomenon-viable, not only absorb dyes excluded by viable cells, but also havea distinctive appearance which makes them recognizable even in thepresence of other dead cells. Thus, it becomes practical to assess aheterogeneous sample, such as blood plasma, or a disaggregated form orextract of the microaggregates described in WO 2007/075440, for thepresence of endothelial cells by subjecting the sample to an agent knownto kill endothelial cells and staining with a suitable dye that rendersthe dead endothelial cells instantly recognizable under a microscopeeven to an unpracticed eye. This permits a rapid and efficient test forcirculating endothelial cells wherein an elevated level of such cells isindicative of traumatic conditions in the subject.

It has also been found, surprisingly, that the nontoxic solvents DMSOand ethanol have themselves antiangiogenic effects and are able to lowerthe levels of vascular endothelial growth factor (VEGF) in cellcultures.

DISCLOSURE OF THE INVENTION

In one aspect, the invention is directed to a method to detect andquantify circulating endothelial cells in human or other vertebratesubjects which comprises subjecting a sample of a bodily fluid to anagent that is toxic to endothelial cells, followed by treating thesample with a dye excluded by viable cells but taken up by non-viablecells, and examining the sample under a microscope to detect, and, ifdesired, count the readily identifiable endothelial cells in the sample.Increased counts of endothelial cells in the circulatory system of thesubject are indicators of a variety of conditions such as diabetes,cardiovascular problems and the like, as well as trauma such as muscularstrain and bruising.

Thus, in this aspect, the invention is directed to a method to detectand optionally quantify circulating endothelial cells in a vertebratesubject which method comprises

(a) contacting a bodily fluid obtained from a subject to be tested withan agent that is cytotoxic to endothelial cells;

(b) treating the cells with a dye that is excluded by viable cells buttaken up by dead cells;

(c) observing the resulting cells under a microscope, and

(d) identifying the endothelial cells in said culture by their smallsize, angular appearance and intense color.

Bodily fluids that are useable include blood, plasma and lymph.

If desired, the number of endothelial cells may be counted and theconcentration of endothelial cells in the circulatory system calculated.The method may also include counterstaining with a stain taken up byviable cells.

The above method can be used to assess the effectiveness of therapeuticagents by evaluating the effect of the agents administered to thesubject on the number of endothelial cells in the circulation of thesubject tested. Animal models are particularly useful in this aspect ofthe invention, though effectiveness of treatments in human or veterinarypatients can be evaluated as well.

It has also been found that because of the distinctive nature of theendothelial cells when treated as described herein, the method of theinvention can be used to assess endothelial cells in tumors bydisaggregating the required microaggregates described in the above-citedPCT publication and evaluating the disaggregated or extracted form forthe quantity or presence of such cells.

In addition, the assay method of the invention has revealed that,surprisingly, the nontoxic substances DMSO and ethanol can potentiatethe effects of antiangiogenesis drugs, since they themselves can exhibitthis effect. Thus, in another aspect, the invention is directed to animproved method to treat conditions characterized by unwantedneovasculature which comprises administering to a subject in need ofsuch treatment an antiangiogenic agent while maintaining blood levels ofethanol and/or DMSO that are nontoxic, but are sufficient to enhance theantiangiogenic effect of the primary drug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the effect of staining of endothelial cells selectivelyaccording to the invention method in comparison with CD31 staining. FIG.1A shows the results of staining a suspension culture with Fast Green,counterstaining with hematoxylin-eosin (H&E) and then counterstainingwith anti-CD31. FIG. 1B shows cells stained only with Fast Green/H&E.FIG. 1C shows the cultured cells treated with bevacizumab and thenstained said Fast Green/H&E.

FIGS. 2A-2H show the ability of various kinase inhibitor drugs to exertan antiangiogenic effect as compared to bevacizumab.

FIGS. 3A-3H show the ability of combinations of bevacizumab with kinaseinhibiting drugs to effect endothelial cell killing.

FIGS. 4A and 4B show the combined effects of imatinib and bevacizumab onendothelial cell killing as determined on a sample containing peripheralblood cells.

FIGS. 5A and 5B show images used to quantify endothelial cells in asample.

FIG. 6 is a graph showing effects of various drugs on endothelial andnon-Hodgkin's Lymphoma (NHL) cells.

FIG. 7 is a graph showing the anti-angiogenic cell effects of ethanoland DMSO.

MODES OF CARRYING OUT THE INVENTION

The invention provides a useful diagnostic tool to assess the generalphysical condition of a subject and to assess the presence of traumaticeffects of, for example, sports injuries or overactivity.

In the context of a general routine physical checkup of a normalsubject, a finding of normal levels of circulating endothelial cellscould spare the subject from additional tests in view of the knownassociation of high counts of endothelial cells with conditions such asdiabetes and cardiovascular problems. Tests designed to detect suchconditions would not be, strictly speaking, necessary in the face ofnormal endothelial cell counts.

On the other hand, a finding of abnormally high endothelial circulatingcells would indicate the need for further assessment. The availabilityof a simple test based on a small blood sample might be attractive inencouraging individuals to evaluate their overall health and such testscould be conducted, not only in single doctor's offices, but also inconvenient locations now served by nurse practitioners or otherparamedical professionals in drug stores and the like.

The method of the invention requires only simple equipment and minimaltraining of personnel to evaluate. This is true because the deadendothelial cells which are able to take-up dyes excluded from viablecells have distinctive appearances. They stain very heavily, they areangular, and they are smaller than contaminating cells. They are roughlyone-third the size of lymphocytes.

While direct observation of the cells and scoring by eye is effective,the method lends itself to quantitation by instrumentation as well.Illustrated below, is the use of ImageJ software to quantitate theresults of the assay.

As used herein, “antiangiogenic agent” or “antiangiogenesis agent”“anti-neovasculature” or “antimicrovascular agent” or “agent cytotoxicto endothelial cells” are used interchangeably to denote agents that areable to kill endothelial cells and thus decrease neovascularization.These agents may interact with VEGF, with VEGF receptors, or with otherfactors that are necessary to promote angiogenesis.

In general, such agents are useful in treating conditions that arecharacterized by unwanted neovasculature. These include cancers, maculardegeneration and other conditions such as benign hypertrophies.

In the assay of the invention, a blood sample is optionally subjected tocentrifugation, gravity sedimentation, differential cell lysis or othertechnique to remove the red blood cells (although, strictly speaking,this is unnecessary) and the resulting plasma treated with an agentknown to be cytotoxic to endothelial cells, such as antibodies directedagainst vascular endothelial growth factor (VEGF) or otherantiangiogenic agent. A convenient choice is Avastin® which is availablecommercially.

After treatment with the antiangiogenesis agent, the sample is treatedwith a suitable dye specific for non-viable cells, such as fast green.The method of the invention is not limited to these particular dyes. Anydyes with these features can be used.

The cells are then spun down to a microscope slide and evaluated.

If desired, the cells on the microscope slide may counterstained with,for example, Wright-Giemsa, H&E or other suitable stain. It is notnecessary to use these particular dyes, but they are convenientembodiments.

The foregoing process is applicable not only to body fluids, but also toextracts of, or disaggregated forms of, microaggregate samples obtainedfrom tumors as described in PCT publication WO 2007/075440. Theneovasculature of the microaggregates appears to provide a scaffoldingfor retaining the three-dimensional structure of the microaggregates,and thus, one method for disaggregation is treatment withantiangiogenesis agents such as bevacizumab. When the scaffold isdestroyed, free endothelial cells are released into the culture medium.The foregoing methods can be applied to assess the presence and/oramount of said cells then released.

This method can also be used to assess the effects of intended therapieson the endothelial cell population by treating a tissue sample, such asthe microaggregate of the above-mentioned PCT publication, with thetreatment to be tested and disaggregating the tissue sample orextracting the cells therefrom, and assessing the effect of thetreatment on the endothelial cell population. In this case, the extractor disaggregation should not, itself, kill the endothelial cells. Thedead endothelial cells can be detected and, if desired, counted asdescribed above by treating them with a dye that is excluded by viablecells but taken up by dead cells and observing the resulting cells undera microscope. The dead endothelial cells are recognized by their smallsize, often angular appearance and often intense and refractile color.

It has also been found that both ethanol and DMSO are able to exertantiangiogenesis effects and are cytotoxic to endothelial cells. Theycan be used in combination with a primary drug that is anantiangiogenesis agent because they are nontoxic when used in lowamounts. Thus, in addition to primary therapy with one or moreantiangiogenesis drugs, a subject may be treated so as to maintainnontoxic levels of alcohol or DMSO in the blood. DMSO would beadministered by, for example, IV injection, while blood alcohol levelscan conveniently be maintained by administering alcohol orally in theform of conventional alcoholic beverages. Breathalyzer tests may be usedto ensure that the appropriate alcohol levels are maintained duringtreatment. As noted in Example 6, below, a patient at the University ofPennsylvania was successfully treated with a combination ofantiangiogenic agents and red wine. In addition, there is anecdotalevidence from the University of Heidelberg where a breast cancer patientwith daily wine consumption of 1.5 l showed a 5-year+ remission ontherapy with the antiangiogenic agent trastuzumab. This is reported byEichbaum, M. H. R., Anticancer Drugs (2005) 16:199-200. It should benoted that this does not represent the combination therapy of theinvention as the patient is a chronic alcoholic and not administered acontrolled protocol.

Preparation A

In the Examples below, where cell culture methods derived from biopsieswere used to conduct the experimental procedures, they were prepared asfollows. The preparation is essentially as set forth in theabove-referenced PCT publication WO 2007/075440.

Fresh biopsies or fluid aspirates are obtained from patients with canceror other illnesses or from normal donors.

Specimens are typically submitted for conduct of the invention methodvia the anatomic pathology laboratories of the submitting hospitals, or,in some cases, directly from the operating room or a surgeon/physicianoffice. Solid tumor specimens (not exposed to fixatives or frozen) areplaced in cold transport medium (CO2-independent medium,InvitroGen/GIBCO, Grand Island, N.Y., supplemented withpenicillin/streptomycin, amphotericin B, insulin/selenium/transferrin,and 10% low endotoxin, heat inactivated fetal bovine serum). Specimensare then placed in sturdy Styrofoam® shipping boxes, containing 350 gmblocks of “blue ice” frozen to minus 20 degrees Celsius. These are thenshipped either by a priority overnight delivery service or via localland courier. Fluid specimens are mixed well to suspend cell clustersand then poured into sterile 500 ml polypropylene transport bottles. Tento fifteen units of heparin sulfate are added per ml of fluid submitted.

Copies of the official histopathology reports from the submittinghospitals should be received.

Solid tumors are minced to pieces smaller than 1 mm (small enough to beaspirated into a standard disposable 10 ml pipette) with high qualitycurved surgical scissors. Medium in which said tumors may have beentransported is reserved, along with the supernatant from the tissuemince Scissor-minced tumor pieces are digested with collagenase/DNase inRPMI-1640 containing antibiotics and 10% fetal calf serum. Specimens aredigested in 50 ml disposable polypropylene centrifuge tubes, assisted bygentle mixing with plastic-coated, magnetic stirring bars over astirring plate. Specimens are thusly mixed until complete grossdigestion has taken place—typically about 2-3 hours for a 1-3 gramspecimen. Cytospin slides are then prepared from all cell fractions(transport medium, supernatant from tissue mince, and enzyme digestate),and stained with fast green-H&E, as described previously (Weisenthal, etal., “A Novel Dye Exclusion Method for Testing in vitro Chemosensitivityof Human Tumors,” Cancer Res. (1983) 43:749-757).

Fluid specimens are centrifuged in their entirety to collect all cellsin the specimen. Cells are then resuspended in the above RPMI-1640-basedmedium and cytospins are prepared as described above.

To normalize the results, “day zero” slides are prepared, depicting thecondition of the cells not exposed to treatment at the beginning of theassays, and “end culture” slides of negative control (non-exposed cells)are also prepared.

To assay for endothelial cells are mixed with, for example, 10%(volume/volume) or, for example, 1% (volume/volume) or intermediatepercentages of antiangiogenic drug containing solution or vehiclecontrol (most typically 0.9% NaCl). Final volume of cell suspension/drugsolution (or vehicle) plated for culture can be 0.12 ml. Culturing is inpolypropylene round bottom, 96-well culture dishes in a humidified 37°C. incubator for a standardized duration of time.

Stock solutions are generally prepared at ten times-100 times thedesired concentrations, aliquotted into single-use, 0.5 ml conicalpolypropylene tubes, and frozen at −70° C. prior to use. Some drugs aremaintained at refrigerator temperature, according to manufacturer'srecommendations.

Cells are cultured with the index concentration of each drug and, ifdesired, with dilutions of the index concentration, where the indexconcentration is determined from training set assays or from theliterature. Negative controls generally consist of 0.9% NaCl, and/or thevehicle in which an antiangiogenic drug is dissolved. Replicate 96-wellplates are tested.

In addition, details of tumor cell isolation, cell culture conditions,slide preparation and staining are described at the website ofWeisenthal.org.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Detection of Endothelial Cells

Using the procedures set forth in Preparation A, a pancreatic carcinoidtumor was visualized after 96 hours in suspension culture in thepresence and absence of 2.5 mg/ml bevacizumab. The cultures were stainedwith the above-described Fast Green/H&E stain/counterstain. FIG. 1Bshows the results in a control culture that has not been treated withbevacizumab. It is apparent that the endothelial cells are barelyvisible. FIG. 1A shows the results of the control culture (not treatedwith bevacizumab) when CD31 staining is used to identify the endothelialcells. Again, they are not particularly conspicuous. FIG. 1C shows theresults when the culture has been treated with bevacizumab, resulting inendothelial cell death. The appearance of the endothelial cells is quitestriking. The characteristic “blueberry pancake” appearance described inthe above-referenced WO 2007/075440 publication is observed.

Example 2 Comparison of Various Chemotherapeutic Agents

The cultures of Example 1 were treated with various candidatechemotherapeutic drugs in comparison to bevacizumab at 2.5 mg/ml andstained as described in Example 1. The various drugs were rated byscoring on a 0+ to 5+ scale for the anti-microvascular effects, with 5+being the highest possible score. As shown in FIGS. 2A-2H, gefitinibscored only 0; sunitinib scored 1+, sorafenib scored 3+ and botherlotinib and bevacizumab scored 4+. The “nib” drugs are kinaseinhibiting drugs.

Further results are tabulated in Table 1 where slides were scored foranti-microvascular effects as described above and also for directantitumor effects as described on the Weisenthal.org website. For thisdetermination, the results provided in Table 1 are 10× multiples of thescoring system of the previous paragraph. The table also shows theconcentration of each drug employed. Bevacizumab was dispensed both in avehicle containing 0.9% NaCl and in a vehicle which resulted in a finalconcentration in the culture medium of 0.5% DMSO/0.5% ethanol. Gefitiniband sunitinib were dispensed in 0.9% NaCl, but erlotinib, sorafenib, andimatinib were dispensed to provide the final 0.5% DMSO/0.5% ethanolconcentration.

TABLE 1 Vehicle Anti- Anti- Vascular Vascular P2 (paired AVS Anti- (AVS− Score Score comparison minus Tumor VAVS)/AT N paired (AVS} (VAVS) AVSVAVS (AT) Score Ratio comparisons (Avg) (Avg) vs VAVS) (Avg) (Avg) (Avg)Bevacizumab 51 24.7 5 <0.0001 19.7 12.9 1.53 2.5 mg/ml DMSO 0.5% + 4012.7 3.5 <0.0001 9.2 3  3.67 EtOH 0.5% Bevacizumab + 37 32.8 11.5<0.0001 21.3 Not scored N/A DMSO/EtOH Cisplatin 37 4.5 3.9 0.37 0.6 59.20.01 3.3 μg/ml Sunitinib 26 9.5 4.6 0.082 4.9 25.8 0.19 8.35 μg/mlGefitinib 32 8.3 3.8 0.0002 4.5 28.5 0.16 22.35 μg/ml Erlotinib 45 16.212 0.0014 4.2 22.5 0.19 89.4 μg/ml Sorafenib 35 12.2 10.8 0.24 1.4 28.70.05 12.5 μg/ml Imatinib 36 13.9 11.1 0.03 2.8 23.6 0.12 12.5 μg/ml

The scores provided in FIG. 2 were multiplied by 10 and averaged to givean anti-vascular score (AVS) shown in column 2; the average AVS for thevehicle alone (VAVS) is shown in column 3 and the level of thestatistical significance of their difference is shown in column 4. TheAVS score was corrected for the effect of vehicle by subtraction asshown in column 5. The antitumor score (AT) is shown in column 6.

Finally, the ratio of AVS (corrected for the effect of vehicle) to theantitumor score is shown in the last column.

As will be apparent from reference to the Table, bevacizumab has arelatively high ratio as compared to the other drugs. It is alsoapparent that the DMSO/EtOH combination (as compared to 0.9% NaCl)itself has a significant anti-microvasculature effect.

Example 3 Synergistic Effects of Combinations

A specimen of poorly differentiated breast cancer was used in theprocedures set forth in the previous examples and tested with variousdrugs and drug combinations as described above. FIGS. 3A-3H show theresults. FIG. 3A shows the appearance after staining with Fast Green/H&Eon a control culture. FIGS. 3B, 3C and 3D show the appearance whentreated with erlotinib, imatinib and bevacizumab alone. None appearedparticularly effective, although bevacizumab was scored as +1.Combinations of bevacizumab with either erlotinib (FIG. 3E) or imatinib(FIG. 3F) showed much better results. FIGS. 3G and 3H are simply greatermagnifications of the results shown in FIG. 3F.

Example 4 Application to Peripheral Blood

Peripheral blood chronic lymphocytic leukemia cells were cultured for 4days in the absence and presence of bevacizumab (2.5 mg/ml) and stainedand counterstained as described above. The results are shown in FIG. 4.FIG. 4A shows the culture without bevacizumab. While endothelial cellscan be seen, they are clearly less visible than in FIG. 4B where thebevacizumab has caused endothelial cell death. They are readily apparentas refractile, angular small cells of smaller dimension than thesurrounding cells. These cells are about one-third the size oflymphocytes and are about 3μ in diameter.

Example 5 Quantification of Cell Count

A lymph node biopsy was obtained from a non-Hodgkin's Lymphoma patientand processed and stained as described above. FIG. 5A is a 200× view ofcells cultured 4 days with bevacizumab. The presence of dead endothelialcells is readily apparent. The cells can also be imaged using publiclyavailable “ImageJ” software available from the National Institutes ofHealth (NIH) to assist quantitation. The image is converted to 8-bitblack and white threshold gated to paint only the dead endothelialcells. Only the painted features were counted and measured by softwareand the resulting image is shown in FIG. 5B.

The same sample was tested against various drug and vehicle combinationswith the results shown in FIG. 6.

The objective, automated ImageJ scores for both individual gatedfeatures and also for total area (originally in pixels squared) isshown. The raw scores for the gated features (putative dead endothelialcells) are normalized for uneven cell distributions by image analyzingeach area twice: first with the detection threshold set to gate only thefeatures of interest (putative dead endothelial cells) and then again todetect all features on the slide (this would be analogous to normalizinggene copy to total DNA in cell extracts). Shown additionally are thesubjective (0+ to 5+) manual anti-vascular scores which were obtainedone month before the installed the ImageJ software was installed. Shownadditionally are the direct antitumor cell effects of each of the drugsand solvents tested.

As shown, 0.9% NaCl as a vehicle had essentially no antitumor oranti-microvascular activity. However, DMSO/EtOH had significantanti-microvascular activity but no direct antitumor activity. In thisparticular sample, doxorubicin and fludarabine had direct antitumoreffects but were relatively ineffective as anti-microvascular agents.Bevacizumab had essentially no direct antitumor effects as expected,while sorafenib, imatinib, and to a lesser extent, erlotinib acteddirectly on tumor cells. Their anti-microvascular effects appearcomparable to those of vehicle alone.

The effects of ethanol and DMSO were further confirmed as shown in FIG.7 using the same sample. These effects were achievable even atrelatively low dosage concentrations of 0.125% of the final medium.

Example 6 Clinical Illustration of the Effect of Blood Alcohol

A patient at the University of Pennsylvania afflicted withadenocarcinoma of the lung which was metastatic to the brain had beenheavily pretreated with chemotherapy, and brain radiation therapywithout significant affects. Subsequently, he was treated with Avastin®(bevacizumab) and Tarceva® (erlotinib) along with 4 days of drinking redwine with a breathalyzer so as to keep the blood alcohol above 0.1%. Asubsequent MRI of his brain showed regression of multiple brain tumors.

Example 7 Detection of Endothelial Cells in the Presence of LymphaticCells Obtained from Multiple Subjects

Lymph samples from five lymphoma patients and two normal volunteers werecultured and stained as described above. In each case, cultures weretreated or not treated with bevacizumab in order to assess the effect ofendothelial cell death on visibility and ease of detection. Thequantitation of results was as described in Example 4 using ImageJsoftware. The results are shown in Table 2.

TABLE 2 Relative Visualization ImageJ Relative ImageJ Diagnosis ReagentFeature Count Feature Area NHL Patient 1 0.9% NaCl 0.32 0.17 NHL Patient1 Bevacizumab 16 23 NHL Patient 2 0.9% NaCl 0 0 NHL Patient 2Bevacizumab 7.8 9.3 NHL Patient 3 0.9% NaCl 8.3 6.5 NHL Patient 3Bevacizumab 54 61 CLL Patient 4 0.9% NaCl 0.84 0.61 CLL Patient 4Bevacizumab 21 34 ALL Patient 5 0.9% NaCl 3.7 4.4 ALL Patient 5Bevacizumab 39 50 Normal Volunteer 6 0.9% NaCl 0.61 0.26 NormalVolunteer 6 Bevacizumab 3.7 1.4 Normal Volunteer 7 0.9% NaCl 2.1 0.8Normal Volunteer 7 Bevacizumab 52 32

As Table 2 shows, the visibility of endothelial cells was greatlyenhanced in all of the lymphoma patients by the addition of bevacizumab.Normal Volunteer 6 had only a small number of endothelial cells in thesample, while Normal Volunteer 7 had high levels. This implies thatVolunteer 6 is free of conditions associated with sloughed endothelialcells where Volunteer 7 is not.

Example 8 Effect of EtOH/DMSO on Levels of Vascular Endothelial GrowthFactor (VEGF) in Cell Cultures

The observed ability of EtOH/DMSO to effect killing ofmicrovascular/endothelial cells may be due to the ability of thesesolvents to reduce the levels of VEGF in cell culture. It is known thattumor cells secrete VEGF and VEGF is needed, for the growth ofneovasculature. Accordingly, a number of tumor cell cultures weretreated with these solvents. The level of VEGF in the cultures wasmeasured by subtracting the optical density at 570 nm from the opticaldensity of 450 nm The results are drawn in Table 3.

TABLE 3 450-570 O.D. 450-570 450-570 Contents of culture well #1 O.D. #2O.D. #3 Std 0 pg/ml 0.058 Std 15.6 0.086 Std 31.2 0.122 Std 62.5 0.235Std 125 0.305 Std 250 0.656 Std 500 1.300 Std 1000 2.180 No cells/RPMI +Serum 0.055 0.057 0.058 No cells/RPMI/No Serum 0.055 0.055 0.056 Nocells/0.9% NaCl 0.056 Pt 1 Cells (P1C; Merkel Tumor, 0.075 0.089 0.07910% DMSO Cryopreserved) P1C + Bev 2.5 mg/ml 0.051 0.052 0.054 P1C + Bev1.25 mg/ml 0.054 0.054 0.059 P1C + EtOH 0.5% + DMSO 0.5% 0.055 0.0550.054 P1C + Cisplatin/Anguidine 0.058 0.062 Pt 2 Cells (P2C; Ovarian,10% 0.065 0.079 0.082 DMSO Cryopreserved) P2C + Bev 2.5 mg/ml 0.0520.056 0.054 P2C + Bev 1.25 mg/ml 0.059 0.055 0.054 P2C + EtOH 0.5% +DMSO 0.5% 0.054 0.054 0.055 P2C + Cisplatin/Anguidine 0.055 0.057 Pt 3Cells (P3C; Breast; Fresh) >3.4 3.34 >3.4 P3C + Bev 2.5 mg/ml 0.0560.059 P3C + Bev 1.25 mg/ml P3C + EtOH 0.5% + DMSO 0.5% P3C +Cisplatin/Anguidine 0.284 0.308 Pt 4 Cells (P4C; NHL, Fresh) 0.051 0.0510.052 P4C + Bev 2.5 mg/ml 0.055 0.055 P4C + Bev 1.25 mg/ml 0.074 0.068P4C + EtOH 0.5% + DMSO 0.5% 0.057 0.052 P4C + Cisplatin/Anguidine 0.0530.051 Pt 5 Cells (P5C; Breast, Fresh) 1.005 1.895 1.09 P5C + Bev 2.5mg/ml 0.053 P5C + Bev 1.25 mg/ml P5C + EtOH 0.5% + DMSO 0.5% P5C +Cisplatin/Anguidine Pt 6 Cells (PC; Breast, Fresh) 2.3 >3.4 P6C + Bev2.5 mg/ml 0.052 P6C + Bev 1.25 mg/ml 0.063 P6C + EtOH 0.5% + DMSO 0.5%0.064 0.097 P6C + Cisplatin/Anguidine 0.098 0.187

Table 3 shows the standard curve for 0-1,000 pg/ml of VEGF with areading of 0.058 for 0 concentration and 2.180 for 1,000 pg/ml. Thetable also shows that samples of medium, medium plus serum, and 0.9%NaCl show 0 VEGF concentration. For tumor samples preserved in 10% DMSO,only very low levels of VEGF were observed. For fresh breast tumorcells, the levels of VEGF were off-scale, but could be brought to 0using bevacizumab as expected. Of particular interest are the cells fromPatient 6, shown at the end of the table. Fresh breast tumor cellsshowed values of VEGF that were very high. The combination of 0.5% EtOHand 0.5% DMSO was able to reduce these levels almost to zero.

1. A method to treat conditions characterized by unwanted neovasculaturein a subject which method comprises administering to a subject in needof such treatment an antiangiogenesis agent combined with maintainingsub-toxic blood levels of ethanol and/or DMSO.
 2. The method of claim 1wherein the antiangiogenesis agent effects endothelial cell death. 3.The method of claim 2 wherein the angiogenesis agent is an inhibitor ofvascular endothelial growth factor (VEGF).
 4. The method of claim 1wherein the antiangiogenesis agent is bevacizumab.
 5. The method ofclaim 1 wherein the DMSO is administered intravenously.
 6. The method ofclaim 3 wherein the ethanol is administered orally.
 7. The method ofclaim 6 wherein the ethanol level is monitored with a breathalyzer.